Clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas9 proteins constitute the CRISPR-Cas9 system. This system provides adaptive immunity against foreign DNA in bacteria (Barrangou, R., et al., “CRISPR provides acquired resistance against viruses in prokaryotes,” Science (2007) 315:1709-1712; Makarova, K. S., et al., “Evolution and classification of the CRISPR-Cas systems,” Nat Rev Microbiol (2011) 9:467-477; Garneau, J. E., et al., “The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA,” Nature (2010) 468:67-71; Sapranauskas, R., et al., “The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli,” Nucleic Acids Res (2011) 39: 9275-9282).
The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner (Gasiunas, G., et al., “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria,” Proc Natl Acad Sci USA (2012) 109: E2579-E2586; Jinek, M., et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science (2012) 337:816-821; Sternberg, S. H., et al., “DNA interrogation by the CRISPR RNA-guided endonuclease Cas9,” Nature (2014) 507:62; Deltcheva, E., et al., “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III,” Nature (2011) 471:602-607), and has been widely used for programmable genome editing in a variety of organisms and model systems (Cong, L., et al., “Multiplex genome engineering using CRISPR/Cas systems,” Science (2013) 339:819-823; Jiang, W., et al., “RNA-guided editing of bacterial genomes using CRISPR-Cas systems,” Nat. Biotechnol. (2013) 31: 233-239; Sander, J. D. & Joung, J. K., “CRISPR-Cas systems for editing, regulating and targeting genomes,” Nature Biotechnol. (2014) 32:347-355).
Jinek, M., et al., (“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science (2012) 337:816-21) showed that in a subset of CRISPR-associated (Cas) systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-part RNA structure, also called “dual-guide,” that directs the CRISPR-associated protein Cas9 to introduce double-stranded breaks in target DNA. At sites complementary to the crRNA-guide (spacer) sequence, the Cas9 HNH nuclease domain cleaves the complementary strand and the Cas9 RuvC-like domain cleaves the non-complementary strand. Dual-crRNA/tracrRNA molecules were engineered into single-chain crRNA/tracrRNA molecules. These single-chain crRNA/tracrRNA directed target sequence-specific Cas9 double-strand DNA cleavage.
However, site-specific nucleases such as Cas9 can introduce double-stranded breaks in DNA in unintended and/or incorrect locations, termed “off-target effects.” Accordingly, methods to reduce or eliminate off-target DNA breaks are highly desirable.
Additionally, DNA double-stranded breaks can be repaired by, for example, non-homologous end joining (NHEJ) or homology-directed repair (HDR). Faithful repair by HDR is inefficient at site-directed breaks of the target nucleic acid because other cellular mechanisms may result in the incorporation of nucleic acids at the site of a double-stranded break or a single-stranded nick. It is apparent there is a clear need to develop novel strategies that mitigate or eliminate off-target genome editing events and increase the efficiency of inserting new material into the sites cut by site-directed nucleases such as Cas9.